Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by the presence of extracellular amyloid plaques composed of amyloid-β (Aβ) surrounded by dystrophic neurites and neurofibrillary tangles. The discovery that certain early-onset familial forms of AD may be caused by an enhanced production of Aβ peptides have led to the hypothesis that amyloidogenic Aβ is intimately involved in the AD pathogenic process.
The generation of Aβ peptides is due to enzymatic cleavage of the larger amyloid precursor protein (APP), which represents a type I membrane protein with a large N-terminal ectodomain and a short intracellular C-terminal domain. Alternative splicing of APP yields eight isoforms with lengths of 677-770 amino acid residues, of which APP695 is the primary transcript in neurons. APP can be processed by two different pathways: (i) nonamyloidogenic processing: Cleavage by α-secretase within the Aβ domain releases a secreted form of APP (sAPPα), thereby precluding the generation of toxic Aβ peptides. Different members of the ADAM protein family (a disintegrin and metalloprotease) have been demonstrated to possess α-secretase activity. (ii) Amyloidogenic processing: This APP processing pathway results in cleavage at the β-secretase site, liberating also a secreted form of APP (sAPPβ), and leading to the generation of a membrane-associated C-terminal fragment named C99. The β-site APP cleaving enzyme 1 (BACE) belongs to the family of aspartyl proteases. Subsequent cleavage of C99 by γ-secretase activity results in the generation of 40-42 residue Aβ peptides, as well as a short intracellular APP fragment named AICD. It has been shown that γ-secretase consists of a complex of different proteins including presenilin-1 (PS1) or presenilin-2 (PS2), as well as nicastrin, anterior pharynx defective (APH-1) and presenilin enhancer 2 (PEN-2).
Mutations in either APP or in the presenilin genes have been linked to familiar, early onset forms of AD (FAD). These cases represent only a minor portion (˜5%), whereas the vast majority of AD cases develop sporadically (sporadic form of AD). Most of the reported APP mutations are located near the secretase cleavage sites and lead to an overproduction of Aβ peptides. Some of these mutations (e.g. the Austrian mutation T714I) as well as a couple of PS1 mutations have a drastic effect on the Aβ42/Aβ40 ratio by strongly increasing Aβ42 production with concomitant suppression of β40 secretion.
Aβ accumulation has an important function in the etiology of AD with its typical clinical symptoms, like memory impairment and changes in personality. Even though it has been observed that Aβ localizes predominantly to abnormal endosomes, multivesicular bodies and within pre- and postsynaptic compartments, the mode of this toxic activity is still a matter of scientific debate.
A promising experimental approach to unravel the role of Aβ in AD pathology has been the generation of transgenic mice overexpressing the amyloid precursor protein (APP). All mouse models mimic the typical AD-like pathological deficits in synaptic transmission, changes in behaviour, differential glutamate responses and deficits in long-term potentiation. In addition, learning deficits and reduced brain volume were evident in transgenic APP models. These characteristics are generally attributed to the overexpression of full-length amyloid precursor protein (APP). Although learning deficits were evident in various APP models, the extent of Aβ-amyloid deposition did not correlate with the behavioural phenotype (Holcomb, L. A., et al. (1999) Behav Genet. 29, 177-185; Wirths, O., et al. (2008) Neurobiology of Aging 29, 891-901). These deficits occurred well before plaque deposition became prominent and may therefore reflect early pathological changes, likely induced by intraneuronal APP/Aβ mistrafficking or intraneuronal Aβ accumulation (reviewed in Bayer, T. A., and Wirths, O. (2008) Genes Brain Behav 7 Suppl 1, 6-11).
N-terminal deletions in general enhance aggregation of β-amyloid peptides in vitro. For example, Kuo et al ((2001) J Biol Chem 276, 12991-12998) have used an integrated chemical and morphological comparison of the Aβ peptides and amyloid plaques present in the brains of APP23 transgenic mice and human AD patients. The lack of posttranslational modifications such as N-terminal degradation, isomerization, racemization, and pyroglutamyl formation of Aβ in APP23 mice provides an explanation for the differences in solubility of Aβ from human AD and transgenic mouse plaques.
Besides Aβ peptides starting with an aspartate at position 1, a variety of different N-truncated Aβ peptides have been identified in AD brains. Ragged peptides including those beginning with phenylalanine at position 4 of Aβ have been reported as early as 1985 by Masters et al. (Proc Natl Acad Sci USA 82, 4245-4249). In addition, other N-terminal truncated peptides have been identified like Aβ5-40/42 (Takeda, K., et al. (2004) Faseb J 18, 1755-1757), Aβ11-40/42 (Liu, K., et al. (2006) Acta Neuropathol (Berl). 112, 163-174; Lee, E. B., et al. (2003) J Biol Chem 278, 4458-4466), and Flemish and Dutch N-terminally truncated amyloid beta peptides (Demeester, N., et al. (2001) Eur J Neurosci 13, 2015-2024). Although Aβ11-40/42 peptides have been observed in neuronal cultures and in the brains of patients with AD, the involvement of these peptides in its pathogenesis remains elusive (Cai, H., et al. (2001) Nat Neurosci 4, 233-234).
Moreover, N-truncated Aβ3(pE) peptides have been identified by several groups from AD brains (Kuo et al., supra), and a new mouse model was generated (TBA2) expressing only N-truncated AβpE3 in neurons (Wirths, O, et al. (2009) Acta Neuropathol 118, :487-96). The TBA2 mice have a glutamate to glutamine substitution at position 3 of Aβ, which is prone to form AβpE3. Aβ3(pE) has a higher aggregation propensity, and stability, and shows an increased toxicity compared to full-length Aβ. This model demonstrated that this peptide is neurotoxic in vivo inducing neuron loss and an associated neurological phenotype. Similarly, US 2009/0098052 A1 and WO 2009/034158 disclose transgenic mice, wherein the transgene encodes various mutant Aβ 3-40 and Aβ 3-42 peptides.
It was demonstrated that in another transgenic mouse model (Tg2576), Aβ peptides lacked a pronounced N-terminal degradation, posttranslational modifications, and cross-linkages that were frequently observed in the compact Aβ peptide deposits found in AD brain. Thus, under in vivo conditions, truncated Aβ molecules appeared to be generated by hydrolysis at multiple sites rather than by post-mortem N-terminal degradation.
Moreover, it was shown that the APP/PS1KI mouse model develops severe learning deficits at six months of age correlating with a CA1 neuron loss and an atrophy of the hippocampus (Casas, C et al. (2004) Am J Pathol 165, 1289-1300; Breyhan, H., et al. (2009) Acta Neuropathol 117, 677-685), together with a drastic reduction of long-term potentiation and disrupted paired pulse facilitation, coinciding with intraneuronal aggregation of N-terminal modified Aβ variants (Breyhan, H., (2009) Acta Neuropathol 117, 677-685). Notably, the APP/PS1KI mouse model exhibits a large heterogeneity of N-truncated Aβx-42 variants (Casas et al., supra). This was accompanied by reduced levels of pre- and post-synaptic markers.
In addition, the inventors have reported that intraneuronal Aβ rather than extracellular plaque pathology correlates with neuron loss in the hippocampus, the frontal cortex and the cholinergic system of APP/PS1KI mice expressing transgenic human mutant APP751 including the Swedish and London mutations on a murine knock-in (KI) Presenilin 1 (PS1) background with two FAD-linked mutations (PS1M233T and PS1L235P).
By using mouse models expressing full-length APP, and after cleavage also C-terminal fragments and Aβ peptides, it is difficult to decipher the pathological function of specific Aβ peptides. In addition, all transgenic lines harbour mutations either from AD families (for example the APP/PS1KI or the 5×FAD mice) or an artificial one as in the TBA2 mice.
All previously reported mouse models with synaptic dysfunction, neuron degeneration and behavioural deficits are based on transgenic constructs with artificial mutations or from inherited familial AD cases. In other words, existing models are based on autosomal dominant mutations in APP and the presenilin genes, which represent models for the rare familial AD (FAD) forms only. Accordingly, there is still a need for a model for the common sporadic form of AD.